Figure 5.

(A,B) Knockdown of PKCζ with siRNA reduces S727 Stat3 phosphorylation in HUVECs during normoxia and following hypoxia-reoxygenation. (A) HUVECs were transfected with control siRNA (si-control) or PKCζ siRNA (si-PKCζ), or with no siRNA (Control), lysed, and immunoblotted. (B) 48 h after transfection HUVECs were exposed to hypoxia for 2h and reoxygenation for 30 min, lysed, and immunoblotted. The experiment was done twice with similar results.

(C,D) Following hypoxia-reoxygenation, PKCζ colocalizes with Stat3 in the cytoplasm, but after phosphorylation of S727, Stat3 is found in the nucleus without PKCζ. (C) HUVECs were exposed to hypoxia for 2 h and reoxygenation for 15 min. Cells were fixed and incubated with rabbit anti-Stat3 and goat anti-PKCζ antibodies with secondary anti-rabbit (red) and anti-goat (green) antibodies, and examined by confocal microscopy (63X). a) Stat3, b) PKCζ, c) nuclei (blue), d) merged image, e) enlargement of cell in box to demonstrate cytoplasmic colocalization of Stat3 and PKCζ. (D) HUVECs under normoxia or exposed to hypoxia for 2h and reoxygenation for 60 min were fixed and incubated with rabbit anti-p-S727 Stat3 and goat anti-PKCζ antibodies and detected by secondary antibodies (p-S727 Stat3, red; PKCζ, green), and examined by confocal microscopy (63X). H-R increases cytoplasmic and nuclear p-S727 Stat3, but there is no colocalization with PKCζ. b) merged image showing lack of colocalization, c) separated image of p-S727 Stat3, d) separated image of PKCζ. The confocal microscopy experiments in C and D were each performed twice with similar results.